Rhodopsin kinase that catalyzes the phosphorylation of rhodopsin with ATP has been purified to a homogeneous state. Properties of the kinase and the intracelluar localization of the phosphorylation reaction have been investigated. The location of the carbohydrate moiety of rhodopsin in the retinal disk membrane has been shown to be exclusively on the inner surface of the membrane. On the other hand, the phosphorylation sites of rhodopsin are located on the outer surface of the disk membrane. These results indicate that rhodopsin is a transmembrane protein.